iPSC lines were reprogrammed from SMARD1 fibroblasts and healthy subjects (from EuroBioBank with appropriate ethical approval) using a nonviral protocol (17 (link)). For genetic correction of the SMARD1 iPSC line and the HEK293 line, 1 × 106 cells were transfected with 5 μg of IGHMBP2 plasmid as previously described (26 ). To differentiate spinal motor neurons, we followed a multistep protocol developed for human embryonic stem cells and iPSCs (25 (link)), using SMARD1 and wild-type iPSCs. Cells were plated with neuronal medium [Dulbecco’s modified Eagle’s medium/F12 (Life Technologies)] supplemented with MEM Non-Essential Amino Acids Solution (Life Technologies), N2 (Life Technologies), and heparin (2 mg/ml; Sigma-Aldrich), to which retinoic acid (0.1 μM; Sigma-Aldrich) was added after 10 days for neural caudalization. Posteriorized neuroectodermal cells were collected at day 17 and suspended for 1 week in the same medium with retinoic acid (0.1 μM) and sonic hedgehog (100 to 200 ng/ml; R&D Systems), followed by the addition of brain-derived neurotrophic factor and glial cell line–derived neurotrophic factor on day 24 (10 ng/ml; PeproTech). A centrifugation gradient was used to maximize motor neuron acquisition (25 (link)). Cells were fixed and immunostained for quantification after culture for at least 24 hours.
Sonic hedgehog
Manufactured by R&D Systems
Sourced in United States
Sonic hedgehog is a recombinant human protein produced in HEK293 cells. It is a signaling molecule involved in the regulation of cell differentiation and patterning during embryonic development.
Automatically generated - may contain errors
Lab products found in correlation
Fibroblast growth factor 2 (fgf2), by R&D Systems (6 mentions)
Retinoic acid, by Merck Group (5 mentions)
Dorsomorphin dihydrochloride, by Bio-Techne (4 mentions)
Putrescine, by Merck Group (4 mentions)
Progesterone, by Merck Group (4 mentions)
Sb 43154, by Bio-Techne (4 mentions)
Noggin, by R&D Systems (4 mentions)
Matrigeltm, by BD (4 mentions)
Y 27632, by Bio-Techne (4 mentions)
Insulin, by Merck Group (4 mentions)
Glucose, by Merck Group (4 mentions)
Heparin, by Merck Group (3 mentions)
Mem non essential amino acids solution, by Thermo Fisher Scientific (3 mentions)
Selenium, by Merck Group (3 mentions)
Apo transferrin, by Merck Group (3 mentions)
Retinoic acid, by R&D Systems (2 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (2 mentions)
Cell culture reagents, by Thermo Fisher Scientific (2 mentions)
Gelatin, by Merck Group (2 mentions)
Valproic acid, by Merck Group (2 mentions)
13 protocols using sonic hedgehog
1
Spinal Motor Neuron Differentiation from SMARD1 iPSCs
2
Differentiation of iPSCs into Spinal Motor Neurons
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iPSC lines were reprogrammed from fALS fibroblasts (L114P SOD1 mutation30 ) and healthy subjects (from Eurobiobank with appropriate ethical approval), using a non-viral protocol, as previously described29 (link). To differentiate iPSCs into spinal MNs, we followed a multistep protocol developed for human embryonic stem cells (ESCs) and iPSCs28 . Cells were plated with neuronal medium (Dulbecco’s modified Eagle’s medium (MEM)/F12 (Life Technologies), supplemented with MEM nonessential amino acids solution (Life Technologies), N2 (Life Technologies), and heparin (2 mg/ml; Sigma- Aldrich), to which retinoic acid (0.1 μM; Sigma-Aldrich) was added after 10 days for neural caudalization. Posteriorized neuroectodermal cells were collected at day 17 and suspended for a week in the same medium with retinoic acid (0.1 μM) and Sonic hedgehog (100–200 ng/ml; R&D Systems), followed by the addition of BDNF and GDNF on day 24 (10 ng/ml; PeproTech). A centrifugation gradient was used to maximize motor neuron acquisition28 . Cells were fixed and immunostained for quantification following seeding in culture for at least 24 h.
3
Stem Cell Differentiation Protocol
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Gelatin, putrescine, selenium, progesterone, apotransferrin, glucose and insulin were obtained from Sigma (Steinheim, Germany). Accutase was from PAA (Pasching, Austria). FGF-2 (basic fibroblast growth factor), noggin and sonic hedgehog were obtained from R&D Systems (Minneapolis, MN, USA). Y-27632, SB-43154 and dorsomorphin dihydrochloride were from Tocris Bioscience (Bristol, UK). MatrigelTM was from BD Biosciences (Massachusetts, USA). All cell culture reagents were from Gibco/Invitrogen (Darmstadt, Germany) unless otherwise specified.
4
Efficient Stem Cell Culture Protocols
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Gelatine, putrescine, sodium selenite, progesterone, apotransferrin, glucose, insulin, ascorbic acid, valproic acid and ICRT3 were obtained from Sigma (Steinheim, Germany). Accutase was from PAA (Pasching, Austria). FGF-2 (basic fibroblast growth factor), FGF-8b, Sonic hedgehog and noggin and were obtained from R&D Systems (Minneapolis, MN, USA). Y-27632, SB-43154, CHIR99021 and dorsomorphin dihydrochloride were from Tocris Bioscience (Bristol, UK). MatrigelTM was from BD Biosciences (Massachusetts, USA). All cell culture reagents were from Gibco/Invitrogen (Darmstadt, Germany) unless otherwise specified.
5
Neural Stem Cell Differentiation
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For in vitro priming, neural stem cells were cultured in neurobasal medium plus B27 (Invitrogen), 0.1 mM 2-mercaptoethanol, 20 ng/ml β-fibroblast growth factor, 1 μg/ml laminin, 5 μg/ml heparin, 10 ng/ml neural growth factor (Invitrogen), 10 ng/ml sonic hedgehog (R&D Systems), 10 μM forskolin (Sigma) and 1 μM retinoic acid (Sigma) for 5 days. For the induction of astrocyte, neural stem cells were cultured in poly-L-lysine coated slides with DMEM medium plus N2 supplement and L-glutamate. All cultures were maintained in a humidified incubator at 37°C and 5% CO2 in air, and half of the growth medium was replenished every third day.
6
Stem Cell Differentiation Protocol
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Gelatine, putrescine, selenium, progesterone, apotransferin, glucose, insulin, valproic acid, and trichostatin A were obtained from Sigma (Steinheim, Germany). Accutase was from PAA (Pasching, Austria). FGF-2 (basic fibroblast growth factor), noggin, and sonic hedgehog were obtained from R&D Systems (Minneapolis, MN, USA). Y-27632, SB-43154, and dorsomorphin dihydrochloride were from Tocris Bioscience (Bristol, UK). MatrigelTM was from BD Biosciences (Massachusetts, USA). All cell culture reagents were from Gibco/Invitrogen (Darmstadt, Germany) unless otherwise specified.
7
Neuronal Culture Reagent Procurement
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DNQX, picrotoxin, MK-801, kynurenic acid, guanosine 5′ triphosphate sodium salt hydrate (GTP), adenosine 5′ triphosphate magnesium salt (ATP), EGTA, Pentobarbital, and chloryl hydrate were purchased from Sigma-Aldrich. K-methylsulfate was purchased from Acros Organics. Tetrodotoxin citrate (TTX) was purchased from AbCam. Neurobiotin tracer was purchased from Vector Laboratories. Cell culture media and Leukemia inhibitory factor were purchased from Life Technologies. LDN-193189, Y27632, and XAV939 were purchased from Stemgent. Fibroblast growth factor-2, Insulin-like growth factor-1, and sonic hedgehog were purchased from R&D Systems. N2 and B27 supplements were purchased from Gibco. Cyclosporin A was purchased from Fisher Scientific.
8
Neural stem cell priming and astrocyte induction
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For in vitro priming, neural stem cells were cultured in neurobasal medium plus B27 (Invitrogen), 0.1 mM 2-mercaptoethanol, 20 ng/mL b-fibroblast growth factor, 1 μg/mL laminin, 5 μg/mL heparin, 10 ng/mL nerve growth factor (Invitrogen), 10 ng/mL sonic hedgehog (R&D Systems), 10 μM forskolin (Sigma), and 1 μM retinoic acid (Sigma) for 5 days. For astrocyte induction, neural stem cells were cultured in poly-l -lysine-coated slides with DMEM medium plus N2 supplement and l -glutamate. All cultures were maintained in a humidified incubator at 37 °C and 5% CO2-humidified atmosphere, and half of the growth medium was replenished every third day.
9
Differentiation of Spinal Motor Neurons from iPSCs
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iPSC lines were generated from fibroblasts obtained from Eurobiobank, see Supplementary Table 2 . The cells were tested for Mycoplasma (MycoAlert kit, Lonza). Spinal motor neurons were differentiated using a protocol developed for human embryonic stem cells and iPSCs56 (link). For the generation of motor neurons from ALS, SMA and control patient iPSCs, cells were plated with neuronal medium composed of DMEM/F12 (Life Technologies), supplemented with MEM nonessential amino acids solution (Life Technologies), N2 (Invitrogen), and heparin (2 mg/ml; Sigma-Aldrich). After 10 days, retinoic acid (RA, 0.1 μM; Sigma-Aldrich) was added for neural caudalization. At day 17, posteriorized neuroectodermal cells were collected. These clusters were then suspended for a week in the same medium with RA (0.1 μM) and sonic hedgehog (100–200 ng/ml; R&D Systems). On day 24, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell-derived neurotrophic factor) (10 ng/ml; PeproTech) were added. To enrich for motor neurons a centrifugation gradient was applied. Motor neurons were subsequently transduced with a lenti-Hb9::GFP construct5 . Cells were fixed and stained for quantification using known neuronal markers (Table 1 ).
10
Generating MGE-like Progenitors from hESCs
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hESC SFTA3 and NKX2.1 control and KO cell lines were maintained and passaged as previously described [25 (link)]. Neural differentiation of ESCs was initiated using the ALK2/3 inhibitor LDN-193189 (Stemgent, 100 nM) and progenitors ventralized to an MGE-like identity by a combination of sonic hedgehog (R&D Systems, 125 ng/mL) and its agonist purmorphamine (Calbiochem, 1 μM) as previously described [26 (link)].
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